Composition and method for culturing potentially regenerative cells and functional tissue-organs in vitro

ABSTRACT

Compositions and methods are provided for culturing in vitro potentially regenerative cells (PRCs) from which functional tissue-organs are regenerated. In one aspect of the invention, a tissue culture medium is provided which comprises at least 50% of water and a sterol compound that is dissolved in a fatty acid-containing oil at a concentration at least 0.1% by weight based on the weight of the oil and added to the water. The culture medium can be used to culture PRCs that are isolated from the body of a mammal to generate functional tissue-organs in vitro with substantially the same physiological structure and function as the corresponding ones existing in vivo and in situ. The cultured PRCs, tissues, and tissue-organs can serve as valuable models for scientific investigation in life sciences, nutraceutical discovery, drug screening, pharmacokinetic studies, medical devices and tissue/organ transplantation.

BACKGROUND OF THE INVENTION Cross Reference to Related Application

This application claims priority to Chinese Patent Application entitled“Potentially Regenerative Cells”, filed: Sep. 27, 2002, Chinese PatentApplication Serial No: 02143546.4. This application is hereinincorporated by reference in its entirety.

1. Field of the Invention

The present invention relates to compositions and methods for culturingcells, tissues, and organs in vitro, and more particularly tocompositions and methods for controlled growth and differentiation ofcells to generate functional tissues and/or organs in vitro, which canbe used as in vitro model systems for nutraceutical and pharmaceuticalstudies and for tissue/organ repair and regeneration in vivo.

2. Description of the Related Art

Since the discovery of genetic materials in the middle of the 19^(th)century, scientists have focused on the biochemistry of the geneticmaterials within a cell, which ultimately led to systematic studies ofmolecular biology of the cell. In the 20^(th) century, after severaldecades of fanatic research on genes, rationality started to return inthis area. Until the end of the 20^(th) century—in the year of 1998 didscientists shift their attention to cell biology and resume the researchon the embryonic development of the human body. Such a developmentalprocess has been described by using the term “stem cells”. It is alsowidely envisioned that embryonic stem cells can be cultured in vitro togenerate various body organs such as hearts, kidneys and livers whichcan then be transplanted autologously back to the body for therapeuticpurposes. It is further imagined embryonic stem cells can also be usedas cell therapy by injecting the cells into the diseased site andcausing the stem cells to repair and regenerate in situ, hopefullycuring the disease eventually. These hopes still remain as fantasiesbecause based on the state of the art it is yet to be seen thatfundamental breakthroughs are made to overcome the frustrationsencountered in identifying and culturing stem cells in vitro.

Generally, the current techniques in cell culture include isolatingcells from the body, putting the cells in a sterile, nutritiousenvironment mimicking that in vivo and at appropriate temperatures andpH levels, and letting the cells to grow while trying to maintain theirstructure and function. The subject of a cell culture includes singlecells and cell clusters.

In the study of medical genetics, the most popular cell lines areperipheral blood lymphocytes (PBL), skin cells, fibroblasts and othercell lines that are able to sustain the growth for a long time in vitro.The advantages of PBL culture are: short operation time, simpletechniques, materials repeated harvestable, etc. These cell line areutilized extensively for chromosome analysis in the clinic. The cellscultured in vitro can be transformed into immortalized cell linesautomatically or in response to external stimuli. Or the immortalizedcell lines can be established directly, which can divide and proliferateforever. The characteristics of the cell lines are 1) aneuploid, and 2)the karyotypes of different cells are not completely uniform. Thesecharacteristics are not as obvious for cell lines established from acell colony.

It is required that the conditions of cell culture in vitro mimic theenvironment of cell growth in vivo. Therefore, non-toxic and sterilecondition is most important of all. Compared to the cells in vivo, thecells lost the ability to defend against microbials and poisons whencells are cultured in vitro. They will die once they are contaminated orwhen self-metabolites are accumulated to a certain level. So theessential condition of cell culture in vitro is to maintain the sterileenvironment and discharge the metabolites.

The temperature is another key factor for tissue culture. The suitabletemperature for human cells is 36.5° C.±0.5° C., and the normalmetabolism of cells will be affected and the cells may die if thetemperature is not within this range. The cultured cells can withstandhypothermia better than hyperthemia: when the temperature is less than39° C., the cell metabolism and the temperature have a direct ratio;human cell will have certain level lesion when it's 39–40° C. for 1hour, but it's repairable; when it's 40–41° C., the lesion willwidespread to almost all cells, only a small half of cells can berepaired; when it's 41–42° C., the lesion is very serious, most of cellwill die, but still it's possible for some cells to be repaired; whenthe cells is under 43° C. for 1 hour, all the cells will die.

Concentrations of gases, mainly oxygen and carbon dioxide, are also oneof the essential conditions for cell culture. Oxygen is in involved inthe tricarboxylic acid cycle, which produces the energy of cellproliferation and all kinds of components for cell growth. When the cellcultured in vitro in an ambient environment, they are incubated underthe atmosphere of 95% air and 5% CO₂.

CO₂ is not only the metabolite of cell, but also the required componentof cell growth and proliferation. The main function of CO₂ ismaintaining the pH of the media. The suitable pH for most cells is7.2–7.4, and cells will be adversely affected at a pH beyond this range.Since the cells can withstand acid better alkali, they tend to growbetter in a slightly acidic environment. Studies have showed that theappropriate pH for primary amniotic cells is 6.8.

The most popular method for pH regulation of the media is to add NaHCO₃into media, because NaHCO₃ can provide CO₂. But since CO₂ is easy toevaporate, so this method is suitable for tissue culture in a closedenvironment. Since HEPES is not toxic to cells and can be used for cellculture, it is advantageously used to maintain the pH of the media underan ambient condition.

The media for cell culture is also very important for cell culture. Itnot only provides the essential materials for cell growth andproliferation, but also forms the environment of cell living. There area lot of kinds of media, they can be divided into semisolid medium andliquid medium based the form of the materials used. If categorizedaccording to the sources of supply, they can be divided as syntheticmedium and natural medium.

Synthetic medium is produced strictly based on the types and quantity ofsubstances required by cell growth. It includes carbohydrates, aminoacids, lipids, inorganic salts, vitamins, minerals, and cell growthfactors. When synthetic media are used alone, the cells are alive, butthey can't proliferate well.

The most common natural medium is serum, especially bovine serum. Thereare a lot of cell growth factors, adhesion-promoting factors and otherlive materials in serum. Used together with synthetic media, the naturalmedium allows the cells to grow and proliferate actively. The commonconcentration is 5–20% serum in the synthetic media.

The cells cultured in vitro can be divided into 2 large groups based ontheir growth characteristics. Group 1 are attached cells: they adhere tothe substrate of the container when they are cultured in vitro, such asamniocytes. Group 2 are suspended cells: they can suspend in the mediumin vitro. The most common attached cells are: fibroblasts, epithelialcells and wandering cells.

All the cells, which have similar shape with fibroblast, can be calledfibroblast-like cells. They got their name because they have a shapesimilar to that of fibroblasts in vivo. They have a fusiform shape oradopt an irregular triangle form on the surface of dishes or flasks;there is a ovum nuclear in the central of cell, cytoplasm expendedoutside for 2–3 cm. The cells originated from mesoderm often grow likethis except for true fibroblasts.

Epithelial cell-like cells are thin and flat with irregular multi-angleson the surface of dishes (or other substrates). When cultured in vitro,the nuclear is round and in the center of the cell, all the cells areconnected to each other tightly to form a single layer. Cells originatedfrom ectoderm or endoderm such as skin, skin derivative or epithelialcells of the digestive tract all belong to this group.

Wandering cells scatter in the media and normally don't join each otherto form clusters. Cytoplasm often stretches out as pseudopodium orapophysis. The cells moved actively and there are a lot of amoeboidmovements, very fast and randomly. These cells are not very stable andsometimes are difficult to be distinguished with other cells. Undercertain conditions, when the density of cells increases, the cells areconnected to each other to form multi-angle shape or fibroblast-likeshape cells such as early stage amniotic cells.

The shapes of cultured cells are different depending on the shape of thesubstrate. The most common one is the cell attaching to the flatsurface. Under microscope, living cells are clear and smooth; thestructure is not so obvious. There are always 1–2 nuclei when the cellis growing normally. When the cell is malfunctional, the profile of cellis manifested more saliently against the background. Sometimes there aregranules or bubbles in cytoplasm, which indicates dysfunction of thecell metabolism.

When PBL are cultured in vitro using the techniques currently available,there are no splitting cells in peripheral blood under the normalcondition, which only occurs when it's abnormal. PHA is a stimulator ofmitosis of human lymphocytes. Under the promotion of PHA, lymphocyteschange into lymphocytoblasts from G₀ phase, and then begin to undergomitosis. By exploiting this character of PHA, abundant actively mitoticcells can be obtained by culturing lymphocytes in a medium containingPHA.

In addition, when preparing chromosomes from tissue culture cells, themost common ones in genetic analysis are the cell lines cultured invitro, most of them being malignant tumor cell lines. These cell linesare attached cell lines, only a small part of them are suspended celllines. They have the following advantages: readily available sources,high rate of cell division and the high resolution of chromosomespecimen. The key to the preparation of tissue cell chromosomes is tounderstand and control the growth development of cell culture in vitro.Only cells in log growth period can have high mitotic rate. So thetiming and dosing of colchicine for the cell is critical for properpreparation of cell chromosome specimen.

Epidermal stem cells have been cultured in vitro in order to generatekeratinocytes for reconstructing autologous or allogenic epidermalsheets that can be used in skin transplantation in wound healing. Toprovide a large amount of keratinocytes, great efforts have been made tocultivate human epidermal stem cells in culture. In preparing epidermalsheets for transplant basal keratinocytes are cultivated in culture toproduce large numbers of progeny. Maintaining these stem cells inculture conditions can be challenging. The quality of the keratinocyteculture system must be carefully monitored by directly demonstrating thepresence of holoclones in culture, periodical clonal analysis of areference strain of keratinocyte both in terms of clonogenic and growthpotential, and monitoring the percentage of aborted colonies.Inappropriate culture conditions can irreversibly accelerate the clonalconversion and can rapidly cause the disappearance of stem cells,rendering the cultured autograft or allograft transplantation useless.

Besides keratinocyte stem cells, other types of stem cells arecultivated in cell culture in an attempt to provide sufficient amount ofcells for tissue repair or other therapeutic use. Embryonic stem (ES)cells can be cultured under proper conditions. Thomson et al.demonstrated that cells from the inner cell mass (ICM) of mammalianblastocysts can be maintained in tissue culture under conditions wherethey can be propagated indefinitely as pluripotent embryonic stem cells.Thomson et al. (1998) Science 282:1145–1147. Primate blastocysts wereisolated from the ICM from the blastocysts and plated on a fibroblastlayer wherein ICM-derived cell masses are formed. The ICM-derived cellmass were removed and dissociated into dissociated cells which werereplated on embryonic feeder cells. The colonies with compact morphologycontaining cells with a high nucleus/cytoplasm ratio, and prominentnucleoli were selected and the cells of the selected colonies were thencultured. In this way, a primate embryonic stem cell line wasestablished. It was observed that after undifferentiated proliferationin vitro for 4 to 5 months, these cells still maintained thedevelopmental potential to form trophoblast and derivatives of all threeembryonic germ layers, including gut epithelium (endoderm); cartilage,bone, smooth muscle, and striated muscle (mesoderm); and neuralepithelium, embryonic ganglia, and stratified squamous epithelium(ectoderm). Thus, it is envisioned that these ES cells can be culturedand regulated under suitable conditions to coax the pluripotent cell todifferentiate into cells of a particular tissue type and/or to formvarious organs in vitro. These cells and organs, wishfully, could beused as transplants to cure various diseases and replace dysfunctionalbody parts.

Although desirable, an in vitro embryonic development process is highlyunpredictable. The conditions under which ES cells differentiate into aspecific type of cell or organ are elusive. It has been found that tomaintain cultured ES cells in their relatively undifferentiated,pluripotent state, they must both express the intrinsic transcriptionfactor Oct4, and constitutively receive the extrinsic signal from thecytokine leukemia inhibitor (LIF). Nichols et al. (1998) Cell95:379–391. Upon withdrawal of LIF, cultured ES cells spontaneouslyaggregate into a mass of cells of various tissue types. Although theprograms of gene expression in these cells somewhat resemble thedifferentiation pathways typical of developing animals, the triggeringof these programs is chaotic.

For successful organ regeneration in the clinic using stem cellscultured in vitro, a major obstacle lies in its way. Stem cells culturedin vitro must be directed to differentiate into site-specific phenotypesonce they are transplanted into the lesion site. Complete deciphering ofthe signal needed for this process is required to guide the design ofthe in vitro tissue culturing conditions. Experimental data obtained byothers in the art show that although multipotent human mesenchymal,mouse neural stem cells, and mouse embryonic stem cells can be grown invitro through the addition of leukemia inhibitory factor (LIF) to theculture medium, mouse ESCs differentiate randomly in vitro and in vivo.Progress in the art has made it possible to induce differentiation ofmouse ESCs into multipotent glial cell precursors in vitro and totransplant them into the brain of myelin-deficient fetal rats. However,question remains unanswered as to whether these multipotent stem cellsharvested from specific tissues or differentiated from ESCs in vitrowill make site-specific tissue when transplanted to injured adulttissues.

Up to date enormous amounts of money and efforts have been made inattempts to repair damaged tissue and dysfunctional organs throughcultivation of stem cells in vitro. However, as discussed above, theculturing process is tedious and requires addition of a delicatelybalanced “cocktail” composed of costly protein growth factors tomaintain proliferation of the stem cells, and the directionaldifferentiation of the stem cells is often difficult to control,depending on multiple factors, and irreproducible.

In summary, the current cell culture techniques developed by others sofar have been shown to be able to maintain the growth and proliferationof cells obtained from the body. It is rarely seen that normal, somaticcells from an adult body can be cultured in vitro to generatephysiologically functional tissue or tissue-organ.

SUMMARY OF THE INVENTION

The present invention provides innovative compositions and methods forculturing in vitro potentially regenerative cells (PRCs) from whichfunctional tissues and organs are regenerated. The invention stems fromthe inventor's novel theory that 1) PRCs are “reserved” copies of cellsproduced during the development of the body; 2) when the body is fullydeveloped, these PRCs exist as regular tissue cells in the adult bodybut maintain the ability or potential to proliferate and differentiatein response to the cues of renewal, repair and regeneration of tissuesand organs in situ; and 3) under suitable regenerative conditions andenvironment, the PRCs are activated to become regenerative stem cellswhich proliferate and directionally differentiate to produce tissuecells needed for tissue/organ renewal, repair and regeneration in vivoand in situ.

Guided by this fundamental theory, a series of in vitro experiments weredesigned and conducted to show that PRCs indeed exist in a wide varietyof tissues and organs in the body, and PRCs isolated from differentsites of the body can be activated and converted into regenerative stemcells in a tissue culture medium comprising the inventive compositionand produce in vitro tissues and/or organs with substantially the samephysiological structure and function as the corresponding ones existingin vivo and in situ. Such tissues and/or organs are herein referred toas “tissue-organs”.

In one aspect of the invention, a cell growth regulator is provided,comprising: a sterol compound that is dissolved in a fattyacid-containing oil at a concentration at least 0.1% by weight based onthe weight of the oil. The sterol compound preferably forms ester withthe fatty acid in the oil under suitable conditions such as at hightemperatures (e.g., >100° C.).

In another aspect of the invention, a tissue culture medium is provided,comprising: at least 50% of water and a sterol compound that isdissolved in a fatty acid-containing oil at a concentration at least0.1% by weight based on the weight of the oil and added to the water.The sterol compound preferably forms ester with the fatty acid in theoil under suitable conditions such as at high temperatures (e.g., >100°C.).

The concentration of the oil in the tissue culture medium preferablyranges from about 1% to 50% by weight, more preferably about 5% to 30%by weight, and most preferably about 10% to 20% by weight.

The concentration of the sterol compound in the oil preferably rangesfrom about 0.5% to 40% by weight, more preferably about 1% to 20% byweight, and most preferably about 2% to 6% by weight.

The fatty acid-containing oil is preferably vegetable oil, morepreferably vegetable oil selected from the group consisting of corn oil,peanut oil, cottonseed oil, rice bran oil, safflower oil, tea tree oil,pine nut oil, macadamia nut oil, camellia seed oil, rose hip oil, sesameoil, olive oil, soybean oil and combinations thereof, and mostpreferably sesame oil.

The fatty-acid is preferably selected from the group consisting ofpalmitic acid, linoleic acid, oleic acid, trans-oleic acid, stearicacid, arachidic acid, and tetracosanoic acid.

According to this embodiment, the culture medium may further comprisewax that is dissolved in the fatty acid-containing oil and added to thewater. The concentration of the wax preferably ranges from about 1% to20% by weight, more preferably from about 2% to 10% by weight, and mostpreferably from about 3% to 6% by weight based on the weight of the oil.

The wax is preferably edible wax, more preferably edible wax selectedfrom the group consisting of beeswax, castorwax, glycowax, andcarnaubawax, and most preferably beeswax.

In yet another aspect of the invention, a method for culturingpotentially regenerative cells in vitro is provided. The methodcomprises: isolating tissue cells or a tissue from a predetermined siteof the body of a mammal; and culturing the isolated tissue cells ortissue in a tissue culture medium under suitable conditions such thatpotentially regenerative cells contained in the isolated tissue cells orcells migrated from the isolated tissue are activated to continuouslyproliferate and differentiate to form a tissue-organ which sharessubstantially the same physiological structure and at least onephysiological function with that of the corresponding tissue in situ andin vivo.

The mammal may be a rodent, a primate or a human, preferably a primate,more preferably a human, and most preferably an adult human. The mammalfrom which the tissue cells or tissues are isolated is preferably alive.Optionally, the mammal may be dead but the tissue cells or tissue arestill viable.

The isolated tissue cells or tissues may be isolated from any site ofthe body of the mammal, for example, the brain, heart, liver, lung,intestine, stomach, kidney, bone marrow, and skin. The isolated tissuecells are not embryonic stem cells, and the isolated tissue is not fromthe blastocyst of the mammal.

Optionally, when a tissue is isolated from the body of the mammal, thetissue is processed in vitro to produce cells which are then isolatedand cultured in the culture medium of the present invention to producethe tissue-organ.

The culture medium may comprise at least 50% of water and a sterolcompound that is dissolved in a fatty acid-containing oil at aconcentration at least 0.1% by weight based on the weight of the oil andadded to the water. The sterol compound preferably forms ester with thefatty acid in the oil under suitable conditions such as at hightemperatures (e.g., >100° C.).

The potentially regenerative cells contained in the isolated tissuecells or tissue may be activated in the culture medium to continuouslyproliferate and differentiate for at least 5 days, preferably for atleast 10 days, more preferably for at least 30 days, and most preferablyfor at least 50 days.

The tissue-organ formed in the culture shares at least one physiologicalfunction with that of the tissue in situ and in vivo, for example, theability to produce molecules with biological activities such asenzymatic activity, signaling and regulatory functions, and the abilityto cause muscle contraction in response to electric current.

According to any of the above embodiments, the sterol compound may be ananimal sterol or a plant sterol (also called phytosterol). Examples ofanimal sterol include cholesterol and all natural or synthesized,isomeric forms and derivatives thereof. Preferably, the sterol compoundis selected from the group consisting of stigmasterol, campesterol,β-sitosterol, chalinosterol, clionasterol, brassicasterol,α-spinasterol, daucosterol, avenasterol, cycloartenol, desmosterol,poriferasterol, and all natural or synthesized, isomeric forms andderivatives thereof. More preferably, the sterol compound is acombination of stigmasterol, β-sitosterol, and campesterol, collectivelyreferred to herein as “sitosterol”.

Optionally, the sterol compound is a combination of stigmasterol andβ-sitosterol.

Also optionally, the sterol compound is a combination of brassicasteroland β-sitosterol.

Also optionally, the sterol compound is a combination of brassicasterol,stigmasterol and β-sitosterol.

Also optionally, the sterol compound is a combination of campesterol,stigmasterol and β-sitosterol.

It is to be understood that modifications to the sterol compound i.e. toinclude side chains also fall within the purview of this invention. Itis also to be understood that this invention is not limited to anyparticular combination of sterols forming a composition.

According to any of the above embodiments, the culture medium mayfurther comprise baicalin dissolved in the oil, preferably at aconcentration ranging from about 0.001 to 2% by weight, more preferablyabout 0.02 to 1% by weight, and most preferably about 0.02% to 0.5% byweight based on the total weight of the oil.

According to any of the above embodiments, the oil in the culture mediumis an oil-extract of huanglian wherein the amount of huangqin is 2–60%by weight based on the total weight of the oil.

Also according to any of the above embodiments, the culture medium mayfurther comprise obaculactone dissolved in the oil, preferably at aconcentration ranging from about 0.001 to 2% by weight, more preferablyabout 0.02 to 1% by weight, and most preferably about 0.02% to 0.5% byweight based on the total weight of the oil.

According to any of the above embodiments, the oil in the culture mediumis an oil-extract of huangbai wherein the amount of huangqin is 2–60% byweight based on the total weight of the oil.

Optionally, the culture medium may further comprise obabenine,preferably at a concentration ranging from about 0.001% to 2% by weight,more preferably about 0.002% to 0.5% by weight, and most preferablyabout 0.003% to 0.1% by weight based on the total weight of the oil.

According to any of the above embodiments, the oil in the culture mediumis an oil-extract of huanglian wherein the amount of huangqin is 2–60%by weight based on the total weight of the oil.

Also optionally, the culture medium may further comprise berberine,preferably at a concentration ranging from about 0.001% to 2% by weight,more preferably about 0.002% to 0.5% by weight, and most preferablyabout 0.003% to 0.1% by weight based on the total weight of the oil.

Also optionally, the culture medium may further comprise narcotoline,preferably at a concentration ranging from about 0.001% to 2% by weight,more preferably about 0.002% to 0.5% by weight, and most preferablyabout 0.003% to 0.1% by weight based on the total weight of the oil.

In a particular embodiment, the oil in the culture medium is anoil-extract of huangqin containing baicalin at a concentration rangingfrom about 0.001 to 2% by weight based on the total weight of the oil,wherein the sterol compound is a phytosterol and the oil is sesame oil.

Also optionally, the oil in the culture medium is an oil-extract ofheshouwu wherein the amount of heshouwu is 2–60% by weight based on thetotal weight of the oil.

Also optionally, the culture medium may further comprise various aminoacids, preferably all 20 natural amino acids (e.g., alanine,asparagines, aspartic acid, cysteine, glutamic acid, glutamine, glycine,phenylalanine, histidine, isoleucine, lysine, leucine, methionine,proline, arginine, serine, threonine, valine, tryptophan, and tyrosine),for providing nutrition support to cell growth. The amino acids may bechemically synthesized or obtained from natural sources. For example, afull spectrum of natural amino acids may be obtained by extractingearthworms, a rich source of protein/amino acids, in oil or alcohol.

In a particular embodiment, the oil in the culture medium is anoil-extract of earthworm wherein the amount of earthworm is 2–60% byweight based on the total weight of the oil.

In yet another aspect of the invention, isolated potentiallyregenerative cells from a predetermined site of the body of a livemammal are provided. The isolated potentially regenerative cells, whencultured in a culture medium under suitable conditions, are capable ofbeing activated to continuously proliferate and differentiate to form atissue-organ which shares substantially the same physiological structureand at least one physiological function with that of the correspondingtissue in situ and in vivo.

The potentially regenerative cells may be isolated from any site of thebody of the mammal such as an adult body of a human, for example, thebrain, heart, liver, lung, intestine, stomach, kidney, bone marrow, andskin. The isolated potentially regenerative cells are not embryonic stemcells, and are not from the blastocyst of the mammal.

The culture medium may comprise at least 50% of water and a sterolcompound that is dissolved in a fatty acid-containing oil at aconcentration at least 0.1% by weight based on the weight of the oil andadded to the water. The sterol compound preferably forms ester with thefatty acid in the oil under suitable conditions such as at hightemperatures (e.g., >100° C.).

The isolated potentially regenerative cells, when cultured in theculture medium, may be able to continuously proliferate anddifferentiate for at least 5 days, preferably for at least 10 days, morepreferably for at least 30 days, and most preferably for at least 50days.

The compositions and methods of the present invention can be utilized togenerate in vitro a large amount of regenerative cells, tissues and/ororgans with normal physiological structure and function. Thesebiological materials can serve as extremely valuable models for basicscientific investigation in every aspect of life sciences, and beutilized in many practical applications such as nutraceutical discovery,drug screening, pharmacokinetic studies, medical devices andtissue/organ transplantation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows human intestinal cells cultured for 55 days in vitro in thepresent of the inventive composition.

FIG. 2 shows active proliferation of human intestinal cells that arePRCs in the culture.

FIG. 3 shows that some of the cloned human intestinal cells adhere toeach other to form primary tissues.

FIG. 4 shows that the primary tissues assemble to form villi of theintestinal mucosa.

FIG. 5 illustrates a dynamic model of the proliferation anddifferentiation of potentially regenerative cells (PRCs) in vivo and insitu.

FIG. 6 shows explants of mouse intestine culture in the presence (leftpanel) and absence (right panel) of the inventive composition.

FIG. 7 shows explants of mouse intestine cultured for 60 days in thepresence (left panel) and absence (right panel) of the inventivecomposition.

FIG. 8 shows that single cells migrated from the explants proliferatedto form clones of cells.

FIG. 9 shows that clones of intestinal cells (left panel) adhere to eachother to form intestinal mucosal tissue (right panel).

FIG. 10 shows the end of an intestinal villus formed in the culture(left panel, its magnified image shown in the right panel).

FIG. 11A shows migration of single cells from explants of mouseintestine.

FIG. 11B shows that the migrated single cells from explants of mouseintestine proliferated in the culture to form primary intestinal tissue.

FIG. 12A shows adhesion of cells to form the basic structure of anintestinal villus.

FIG. 12B shows that a complete basic structure of an intestinal villusis formed in the culture.

FIG. 13 shows that an intestinal villus is cloned in the culture.

FIG. 14 compares the basic structure of the cloned intestinal villusaccording the present invention (right panel) with that obtained from atissue section of the intestine of a mouse fetus (left panel).

FIG. 15 compares the structure of the cloned intestinal villus accordingthe present invention (right panel, its magnified image shown in theupper left panel) with that obtained from a tissue section of theintestine of a mouse fetus (lower left panel).

FIG. 16A shows proliferation and differentiation of progenitor cells(upper left panel) in mouse bone marrow into small colonies (lower leftpanel) which evolved into bone marrow tissue (lower right panel)gradually. Control cells are shown in the upper right panel.

FIG. 16A shows proliferation and differentiation of progenitor cells(upper left panel) in mouse bone marrow into small colonies (lower leftpanel) which evolved into bone marrow tissue gradually (moving from thelower to the upper right panel).

FIG. 16B shows progenitor cells (upper left panel) in mouse bone marrowcultured in the absence of the inventive composition only yielded moreand more fibroblasts (moving from the lower left panel to the lowerright panel and then to the upper right panel).

FIG. 17A shows regenerated nerve tissues in the presence (left side ofthe upper and lower panels) the inventive composition and degeneratednerve tissue in the absence of the inventive composition (right side ofthe upper and lower panels).

FIG. 17B shows the structure of regenerated nerve tissues in thepresence of the inventive composition (upper left panel, its HE stainedimage shown in the lower left panel) and the structure of degeneratednerve tissue in the absence of the inventive composition (upper rightpanel, its HE stained image shown in the lower right panel).

FIG. 18 shows cloned pancreatic tissue after culture in the presence ofthe inventive composition for 65 (upper left panel) and 92 days (lowerleft panel), and necrosis of pancreatic cells in the absence of theinventive composition for 65 (upper right panel) and 92 days (lowerright panel).

FIG. 19 shows levels of amylopsin (left panel) and insulin (right panel)in the culture medium of pancreatic tissue in the presence and absenceof the inventive composition.

FIG. 20 shows the cloning of renal structure units (lower left panel andupper right panel) from renal cortical cells (upper left panel). Renalcortical cells died in the control group (lower right panel).

FIG. 21A shows that single cells (upper left panel) isolated from humanhair follicles proliferated and differentiated in vitro to form colonies(lower left panel), the basic structure of a hair follicle (lower rightpanel) and a completely cloned hair follicle.

FIG. 21B shows a magnified image of the cloned hair follicle withcollagenous fiber growing from the hair follicle.

FIG. 22 shows that mouse cardiomuscular cells (upper left panel) werecultured in vitro to gradually form cardiomuscular tissue (moving fromthe lower left panel to the lower right panel and then to the upperright panel).

FIG. 23 shows that rat thymocytes (upper left panel) were cultured invitro to gradually form thymic tissue (moving from the lower left panelto the lower right panel) while thymocytes in the control group died(upper right panel).

FIG. 24 shows that rat hepatocytes (upper left panel) were cultured invitro to gradually form hepatic lobules (the lower left panel) and livertissue (lower right panel) while hepatoytes in the control group died(upper right panel).

FIG. 25 shows treatment of acute gastric ulcer of mice with anembodiment of the inventive composition (right panel: test group; leftpanel: control group).

FIG. 26 shows treatment of gastric ulcer of a patient mice with anembodiment of the inventive composition (left panel: before treatment;right panel: after treatment).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides innovative compositions and methods forculturing potentially regenerative cells (PRCs) from which functionaltissues and organs are regenerated in vitro. Also provided are isolatedcells that possess regenerative potential and are capable of beingcultured to generate in vitro a functional tissue or organ havingsubstantially the same physiological structure and function as thecorresponding tissue or organ residing in vivo and in situ. Thecomposition and methods of the present invention can be utilized togenerate in vitro a large amount of PRCs, tissues and/or organs withnormal physiological structure and function. These biological materialscan serve as extremely valuable models for basic scientificinvestigation in every aspect of life sciences, and be utilized in manypractical applications such as nutraceutical discovery, drug screening,pharmacokinetic studies, medical devices and tissue/organtransplantation.

The invention stems from the inventor's novel theory that 1) PRCs are“reserved” copies of cells produced during the development of the body;2) when the body is fully developed, these PRCs exist as regular tissuecells in the adult body but maintain the ability or potential toproliferate and differentiate in response to the cues of renewal, repairand regeneration of tissues and organs in situ; and 3) under suitableregenerative conditions and environment, the PRCs are activated tobecome regenerative stem cells which proliferate and directionallydifferentiate to produce tissue cells needed for tissue/organ renewal,repair and regeneration in vivo and in situ.

Guided by this fundamental theory, a series of in vitro experiments weredesigned and conducted to show that PRCs indeed exist in a wide varietyof tissues and organs in the body, and PRCs isolated from differentsites of the body can be activated and converted into regenerative stemcells in a tissue culture medium comprising the inventive compositionand produce in vitro a tissue and/or organ with substantially the samephysiological structure and function as the corresponding one existingin vivo and in situ.

During the in vitro experiments it was observed that when somatic tissuecells were isolated from an adult and cultured in vitro (as isolatedcells or explants) in the inventive culture medium, there were somecells that appeared to share the same morphology as the rest of thecells but possessed an unusual potential to be activated to behave likeregenerative stem cells. Initially, these cells remained dormant in theculture but was activated within a few days of culture to manifest theability of a stem cell—to not only constantly proliferate but alsodirectionally differentiate to generate a tissues and/or organ byfollowing a lineage specific to the site of the body where they wereoriginally isolated from. Tissue cells have such characteristics aretermed “PRCs”. While not wishing to be bound to the theory, the inventorbelieves that PRCs differ from classic stem cells in the severalaspects.

Although the definition of stem cells still remains controversial andsubject to changes in the techniques of identification in the field, aclassic definition of a stem cell is that a stem cell should have thefollowing properties: 1) It is not itself terminally differentiated,i.e., not at the end of a pathway of differentiation; 2) It can dividewithout limit or at least for the life time of the animal; and 3) Whenit divides, each daughter cell can either remain a stem cell, or embarkon a course leading irreversibly to terminal differentiation. InMolecular Biology of the Cell, Alberts et al., eds, 3^(rd) ed. (1994),pp. 1155–1156, Garland Publishing Inc., New York and London. Accordingto this definition, stem cells isolated from human tissue, such as theembryonic stem cells isolated from the inner cell mass of humanblastocysts, hematopoietic stem cells from the blood, and epidermal stemcells from the basal layer of the skin are typical stem cells. Recently,adult stem cells (ASCs) have been discovered in the liver, pancreas, andcentral nervous system. See review by Fuchs and Segre (2000) Cell100:143–155. The locations of ASCs have been searched extensively andspeculated by others to be residing in specific “niches”.

The inventor believes that PRCs exist in virtually every tissue andorgan of the body and may not need specific “niches” to be tucked away.As shown in the Example section below, in all of the tissues isolatedfrom mammals PRCs were identified and able to be activated, proliferatecontinuously and directionally differentiate to produce various tissuecells that eventually form a functional “tissue-organ” (See definitionbelow) in vitro.

The inventor also believes that PRCs are duplicates of cells producedduring the body's development from an embryo to a fully developed adult.PRCs tend to stay dormant in the body whereas typical stem cells aremore dynamic in nature, i.e., undergo constantly renewal at differentrates depending on where they reside, such as the constant renewal ofepidermal stem cells from the basal layer of the skin. When there is aneed for repair and regeneration, for example, a tissue/organ damageoccurring in the body, PRCs can be mobilized to start the repair job ifsuitable regenerative conditions are provided. Once activated, PRCsappear to behave like typical stem cells: constantly proliferate anddifferentiate to produce large numbers of tissue cells. Under theenvironment provided by the inventive culture medium, these activatedPRCs do not differentiate chaotically. Instead, the differentiationfollows a lineage specific to the site of the body where the PRCs areoriginally isolated from. The activated PRCs that are going through thisdynamic process are herein termed as “regenerative stem cells”. It isnoted that although PRCs can be precursors of regenerative stem cells,typical stem cells (adult or embryonic) participating the regenerationprocess also fall within the scope of the regenerative stem cells.

A dynamic model of PRCs is hypothesized and illustrated in FIG. 5. PRCsexist in every tissue or organ of the body (e.g., the heart) and can beoriginated from asymmetric division of a proliferating cell. One of thetwo daughter cells (cell B, e.g., a heart muscle cell, FIG. 5) iscommitted to further proliferation for the development of the organwhile the other (cell A, FIG. 5) stops proliferation and remainsdormant. Cell A may be undifferentiated, partially differentiated orpermanently differentiated. However, when the body is injured ordysfunctional, in response to the cues of renewal, repair andregeneration of tissues and organs the dormant cells (cell A) in situare activated. Under suitable regenerative conditions and environment,these cells (cell A) start to proliferate and directionallydifferentiate to produce tissue cells needed for tissue/organ renewal,repair and regeneration in vivo and in situ. These cells (cell A, FIG.5) with the potential to proliferate and directionally differentiate forthe body's repair and regeneration are the PRCs.

The inventor discovered that the PRCs, when isolated from any organ ortissue of the body and cultured under suitable regenerative conditionsin vitro, can proliferate and directionally differentiate into atissue-organ with substantially the same physiological structure andfunction as that in vivo and in situ. A tissue-organ is herein definedas a unit in any organ or tissue of the body that plays not onlystructural but also functional role(s) in maintaining the vitality ofthe body. The tissue-organ is a tissue or a composite of severaltissues, which can be composed of a single or several types of cells.While not wishing to be bound by the theory, the inventor believes thatthere may be about 206 tissue-organs in a human body, which areclassified according to the definition of “tissue-organ” providedherein.

For example, the intestinal villi is such a tissue-organ. It is knownthat intestinal villi are composed of epithelial cells, goblet cells,Paneth cells, and endocrinal cells. While these cells adhere to eachother to form the distinct brush-like structure of intestinal villi,goblet cells in the villi function to secret mucus which is essentialfor protecting the intestine from the harmful effects of exogenousmaterials such as food and drinks, as well as for digesting theseforeign materials in order to provide nutrients to the body.

Similarly, physiologically functional units in the body, such as hairfollicles, pancreatic ducts, pancreatic islets, bone marrow, andganglia, are examples of tissue-organs according to the presentinvention. As will be shown in detail in the EXAMPLE section below, PRCsisolated from various organ or tissue of the body could be cultured invitro under suitable conditions to proliferate and directionallydifferentiate into a tissue-organ corresponding to the one from whichthe PRCs were originally isolated.

The discovery of the PRCs and their potential to develop into functionaltissue-organs in vitro under suitable regenerative conditions hasprofound significance in both theory and practice.

The mainstream theory in the art is that once an organ of a fullydeveloped adult body is injured or dysfunctional, it is almostimpossible to rely on the body itself to completely heal. The popularremedial approach is to replace the failing organ and damaged tissuewith organ transplantation and implantation of bionic device. The majordrawbacks to organ transplantation are donor shortages andimmunosuppressive side effects. The drawback to the approach ofimplantation of bionic device is the inability to manufacture artificialmaterials that duplicate the durability, strength, form, function, andbiocompatibility of natural tissues.

The inventor believes that a fully developed adult body can regenerateitself through activation of PRCs residing in every organ or tissue ofthe body if suitable regenerative conditions are provided. Thisregeneration requires active intervention by delivering compounds orcompositions to the damaged tissue or organ where they function toactivate the PRCs and maintain the proliferation and directionaldifferentiation of the PRCs in vivo and in situ. The compounds orcompositions with such functions are herein defined as “vitalsubstances”. As a result, the newly generated cells and tissues from thebody itself serve to repair the dysfunctional tissue or organ withoutgoing through transplantation.

According to the present invention, compositions and methods areprovided for isolating and culturing PRCs in vitro. The cultured PRCscan develop into functional a tissue-organ with substantially the samephysiological structure and function as the one from which the PRCs wereoriginally isolated. The cultured PRCs and tissue-organ can serve as anin vitro model for studies of cell/tissue/organ development, validationof clinical results, and screening for therapeutic or nutritionalmaterials for treating disease or maintaining the vitality of the body.

In one aspect of the invention, a tissue culture medium is provided,comprising: at least 50% of water and a sterol compound that isdissolved in a fatty acid-containing oil at a concentration at least0.1% by weight based on the weight of the oil and added to the water.The sterol compound preferably forms ester with the fatty acid in theoil under suitable conditions such as at high temperatures (e.g., >100°C.).

The concentration of the sterol compound in the oil preferably rangesfrom about 0.5% to 40% by weight, more preferably about 1% to 20% byweight, and most preferably about 2% to 6% by weight.

The fatty acid-containing oil is preferably vegetable oil, morepreferably vegetable oil selected from the group consisting of corn oil,peanut oil, cottonseed oil, rice bran oil, safflower oil, tea tree oil,pine nut oil, macadamia nut oil, camellia seed oil, rose hip oil, sesameoil, olive oil, soybean oil and combinations thereof, and mostpreferably sesame oil.

The fatty-acid is preferably selected from the group consisting ofpalmitic acid, linoleic acid, oleic acid, trans-oleic acid, stearicacid, arachidic acid, and tetracosanoic acid.

According to this embodiment, the culture medium may further comprisewax that is dissolved in the fatty acid-containing oil and added to thewater. The concentration of the wax preferably ranges from about 1% to20% by weight, more preferably from about 2% to 10% by weight, and mostpreferably from about 3% to 6% by weight based on the weight of the oil.

The wax is preferably edible wax, more preferably edible wax selectedfrom the group consisting of beeswax, castorwax, glycowax, andcarnaubawax, and most preferably beeswax.

Beeswax has long been used as an excipient for manufacturing drugs forexternal use. In traditional Chinese medicine, beeswax is a drug fordetoxication, granulation promotion, for relieving pain and cardialgiaand treating diarrhea, pus and bloody stool, threatened abortion withvaginal bleeding, septicemia, refractory ulcer and thermal injury (“ADictionary of Chinese Materia Medica”, in Chinese, “Zhong Yao Da CiDian”, Science and Technology Press, Shanghai, 1986, page 2581).

The constituents of beeswax can be grouped into four categories, i.e.,esters, free acids, free alcohols and paraffins. Beeswax also containstrace amount of essential oil and pigment. Among the esters, there aremyricyl palmitate, myricyl cerotate, and myricyl hypogaeate. In freeacids, there are cerotic acid, lignoceric acid, montanic acid, melissicacid, psyllic acid, hypogaeic acid and neocerotic acid. Among freealcohols, there are n-octacosanol and myricyl alcohol and in theparaffins, pentacosane, heptacosane, nonacosane and hentriacontane, andan olefin called melene. An aromatic substance called cerolein is alsofound in beeswax.

While not wishing to be bound to the theory, the inventor believes thatthe wax in the culture medium may provide structural support to thesterol compound dissolved in oil and allow the sterol compound to beslowly released to the medium. In addition, the wax may act like asponge to absorb metabolic waste generated by the cells in the culture,further enhancing the active proliferation of the cells.

Optionally, the culture medium may further comprise propolis at aconcentration ranging from about 0.1% to 30% by weight, more preferablyfrom about 1% to 20% by weight, and most preferably from about 5% to 10%by weight based on the total weight of the oil.

Propolis is known as a sticky, gum-like substance which is used to buildthe beehives. In intact propolis a variety of trace ingredients in formof a homogenous mixture with resins, beeswax, essential oils and pollensas predominant ingredients, as well as other ingredients such asflavonoids and phenol carboxylic acids. Natural propolis hardlydissolves in water and has a peculiar odor. Propolis can be preparedfrom beehives by extraction with organic solvents such as ethonol, etherand chloroform.

In another aspect of the invention, a method for culturing potentiallyregenerative cells in vitro is provided. The method comprises: isolatingtissue cells or tissues from a predetermined site of the body of a livemammal; and culturing the isolated tissue cells or tissues a culturemedium under suitable conditions such that potentially regenerativecells contained in the isolated tissue cells or cells migrated from theisolated tissues are activated to continuously proliferate anddifferentiate to form a tissue-organ which shares substantially the samephysiological structure and at least one physiological function withthat of the corresponding tissue in situ and in vivo.

The mammal may be a rodent, a primate or a human, preferably a primate,more preferably a human, and most preferably an adult human. Theisolated tissue cells or tissues may be isolated from any site of thebody of the mammal, for example, the brain, heart, liver, lung,intestine, stomach, kidney, bone marrow, and skin. The isolated tissuecells are not embryonic stem cells, and the isolated tissue is not fromthe blastocyst of the mammal.

The culture medium may comprise at least 50% of water and a sterolcompound that is dissolved in a fatty acid-containing oil at aconcentration at least 0.1% by weight based on the weight of the oil andadded to the water. The sterol compound preferably forms ester with thefatty acid in the oil under suitable conditions such as at hightemperatures (e.g., >100° C.).

The potentially regenerative cells contained in the isolated tissuecells or tissue may be activated in the culture medium to continuouslyproliferate and differentiate for at least 5 days, preferably for atleast 10 days, more preferably for at least 30 days, and most preferablyfor at least 50 days.

The tissue-organ formed in the culture shares at least one physiologicalfunction with that of the tissue in situ and in vivo, for example, theability to produce molecules with biological activities such asenzymatic activity, signaling and regulatory functions, and the abilityto cause muscle contraction in response to electric current.

For example, a viable explant of tissue from a specific organ of ananimal (e.g., a mouse) can be obtained by surgery. The explant is washedwith proper buffer (e.g., PBS containing antibiotics) under sterileconditions, cut into small pieces (e.g., 1 mm×1 mm) and placed inculture plates with suitable sizes (e.g., 6-well, 24-well and 96-wellculture plates), preferably with tissue pieces separated from each otherin the plates. A regular culture medium (e.g., 0.5 ml of MEM) can beadded to the culture, for example, from the edge of the plate in ordernot to disturb the tissue pieces, and the tissue culture is incubatedunder proper conditions (e.g., in a 37° C., 5% CO₂ incubator) for 1–2hr. The cell growth regulator of the present invention is then added tothe plates, the mixture of the cell growth regulator and the regularculture medium constituting an embodiment of the tissue culture mediumof the present invention. Under the regenerative conditions provided bythe tissue culture medium of the present invention, the PRCs containedin the tissue pieces can migrate from the original tissue, activelyproliferate and directional differentiate to generate new tissue andtissue-organ with substantially the same physiological structure andfunction as the one existing in vivo and in situ.

Optionally, the tissue explants may be homogenized and digested withproper enzymes (e.g., trypsin and collagenase) to produce single cells.The cells can be separated from the digestive solution and undigestedtissues, washed and incubated in the tissue culture medium of thepresent invention under conditions (e.g., 37° C., 5% CO₂). Under theregenerative conditions provided by the tissue culture medium of thepresent invention, the PRCs among the single cells isolated from thetissue explants can actively proliferate and directional differentiateto generate new tissue and tissue-organ with substantially the samephysiological structure and function as the one existing in vivo and insitu.

According to any of the above embodiments, the sterol compound may be ananimal sterol or a plant sterol (also called phytosterol). Examples ofanimal sterol include cholesterol and all natural or synthesized,isomeric forms and derivatives thereof. Preferably, the sterol compoundis selected from the group consisting of stigmasterol, campesterol,β-sitosterol, chalinosterol, clionasterol, brassicasterol,α-spinasterol, daucosterol, avenasterol, cycloartenol, desmosterol,poriferasterol, and all natural or synthesized, isomeric forms andderivatives thereof. More preferably, the sterol compound is acombination of stigmasterol, β-sitosterol, and campesterol, collectivelyreferred to herein as “sitosterol”.

Optionally, the sterol compound is a combination of stigmasterol andβ-sitosterol.

Also optionally, the sterol compound is a combination of brassicasteroland β-sitosterol.

Also optionally, the sterol compound is a combination of brassicasterol,stigmasterol and β-sitosterol.

Also optionally, the sterol compound is a combination of campesterol,stigmasterol and β-sitosterol.

It is to be understood that modifications to the sterol compound i.e. toinclude side chains also fall within the purview of this invention. Itis also to be understood that this invention is not limited to anyparticular combination of sterols forming a composition.

It is to be understood that modifications to the sterol compound i.e. toinclude side chains also fall within the purview of this invention. Itis also to be understood that this invention is not limited to anyparticular combination of sterols forming a composition. In other words,any sterol compound alone or in combination with other sterol compoundin varying ratios as required depending on the nature of the ultimateformulation fall with the purview of this invention.

The sterol compound for use in this invention may be procured from avariety of natural sources. For example, phytosterol may be obtainedfrom the processing of plant oils (including aquatic plants) such ascorn oil, wheat germ oil, soy extract, rice extract, rice bran, rapeseedoil, sesame oil, and other vegetable oils, and fish oil. Withoutlimiting the generality of the foregoing, it is to be understood thatthere are other sources of phytosterols such as marine animals fromwhich the composition of the present invention may be prepared. Forexample, phytosterols may be prepared from vegetable oil sludge usingsolvents such as methanol. Alternatively, phytosterols may be obtainedfrom tall oil pitch or soap, by-products of the forestry practice.

Although not wishing to be bound by the theory as to the mechanism ofaction of the sterol compound in activation of the PRCs, the inventorbelieves that the sterol compound may play important roles in inducingmorphogenesis of the cells by changing the fluidity and permeability ofthe cell membrane. As a result, many cell membrane-associated proteinssuch as kinases and phosphotases may be activated to stimulate cellgrowth. It is also plausible that dormant PRCs may be activated due tomorphogenic changes in the membrane. Further, differentiated adulttissue cells may also be induced to undergo transformation into anon-differentiated phenotype, i.e., the process called“dedifferentiation”. With the change of permeability of the cellmembrane, other mitogens and regulatory molecules may be more readilyuptaken by the cells so as to stimulate a balanced growth of a widevariety of cells needed for physiological tissue repair and functionalorgan regeneration. Moreover, expression and phosphorylation of celladhesion molecules (CAMs) may be stimulated, presumably due toactivation of membrane-bound proteins during the morphogenesis process,thus further enhancing association of cognate cells to form a specifictissue, and assembly of cognate tissues to form a functionaltissue-organ in the cell culture.

According to any of the above embodiments, the culture medium mayfurther comprise baicalin dissolved in the oil, preferably at aconcentration ranging from about 0.001 to 2% by weight, more preferablyabout 0.02 to 1% by weight, and most preferably about 0.02% to 0.5% byweight based on the total weight of the oil.

Baicalin may have anti-inflammatory effects on the cells, which helpsproviding a low inflammation environment for the cloning of tissue-organin the culture. It might also be possible that baicalin might bind tocell membrane receptors for polysaccharides such as selectin and furtherpromote cell adhesion.

Baicalin may be obtained by extracting huangqin (Scutellaria baicalensisGeorgi) in oil, alcohol or other organic solvent, preferably in oil attemperature higher than 100° C., more preferably between about 120–200°C., and most preferably between about 160–180° C. Preferably, the rootof huangqin is used and may be obtained from the plant selected from oneor more members of the group of Scutellaria viscidula Bge, Scutellariaamoena C. H. Wright, Scutellaria rehderiana Diels, Scutellariaikonnikovii Juz, Scutellaria likiangensis Diels and Scutellariahypericifolia Levl of Labiatae Family. Dictionary of Chinese MateriaMedica, Shanghai Science and Technology Press, 1988, pages 2017 to 2021.

According to any of the above embodiments, the oil in the culture mediumis an oil-extract of huangqin wherein the amount of huangqin is 2–60% byweight based on the total weight of the oil.

Also according to any of the above embodiments, the culture medium mayfurther comprise obaculactone dissolved in the oil, preferably at aconcentration ranging from about 0.001 to 2% by weight, more preferablyabout 0.02 to 1% by weight, and most preferably about 0.02% to 0.5% byweight based on the total weight of the oil.

Obaculactone is also called limonaic acid and may be obtained byextracting huangbai (Phellodendron amurense Rupr) in oil, alcohol orother organic solvent, preferably in oil at temperature higher than 100°C., more preferably between about 120–200° C., and most preferablybetween about 160–180° C. Alternatively, obaculactone may also beobtained by extracting huangbai in alcohol such as ethanol. Preferably,the bark of huangbai is used and may be obtained from the plant selectedfrom one or more members of the group of Phellodendron chinense Schneid,Plellodendron chinense Scheid var. glabriusculum Schneid, Phellodendronchinense Schneid var. omeiense Huang, Phellodendron Schneid var.yunnanense Huang and Phellodendron chinense Schneid var. falcutum Huang.A Dictionary of Chinese Materia Medica, Shanghai Science and TechnologyPress, 1988, pages 2031 to 2035.

According to any of the above embodiments, the oil in the culture mediumis an oil-extract of huangbai wherein the amount of huangqin is 2–60% byweight based on the total weight of the oil.

Optionally, the culture medium may further comprise obabenine,preferably at a concentration ranging from about 0.001% to 2% by weight,more preferably about 0.002% to 0.5% by weight, and most preferablyabout 0.003% to 0.1% by weight based on the total weight of the oil.

Optionally, the inventive composition may further comprise obabenine,preferably at a concentration ranging from about 0.001% to 2% by weight,more preferably about 0.002% to 0.5% by weight, and most preferablyabout 0.003% to 0.1% by weight.

Obabenine may be obtained by extracting huangqin, huangbai, and/orhuanglian (coptis chinensis Franch) in oil, alcohol or other organicsolvent. Root of huanglian is preferably used. Huanglian may be selectedone or more from the group of Coptis deltoidea C. Y. Cheng et Hsiao,Coptis omeiensis (Chen) C. Y. Cheng, and Coptis teetoides C. Y. Cheng ofRanunculaceae Family. A Dictionary of Chinese Materia Medica, ShanghaiScience and Technology Press, 1988, pages 2022 to 2030.

According to any of the above embodiments, the oil in the culture mediumis an oil-extract of huanglian wherein the amount of huangqin is 2–60%by weight based on the total weight of the oil.

Also optionally, the culture medium may further comprise berberine,preferably at a concentration ranging from about 0.001% to 2% by weight,more preferably about 0.002% to 0.5% by weight, and most preferablyabout 0.003% to 0.1% by weight based on the total weight of the oil.

Also optionally, the culture medium may further comprise narcotoline,preferably at a concentration ranging from about 0.001% to 2% by weight,more preferably about 0.002% to 0.5% by weight, and most preferablyabout 0.003% to 0.1% by weight based on the total weight of the oil.

In a particular embodiment, the oil in the culture medium is anoil-extract of huangqin containing baicalin at a concentration rangingfrom about 0.001 to 2% by weight based on the total weight of the oil,wherein the sterol compound is a phytosterol and the oil is sesame oil.

Also optionally, the oil in the culture medium is an oil-extract ofheshouwu wherein the amount of heshouwu is 2–60% by weight based on thetotal weight of the oil.

Also optionally, the inventive composition may further comprise anextract of heshouwu (Polygonum multiflorum Thunb which belongs to thefamily of Polygonacea), preferably the root tuber of heshouwu (Radixpolygoni multiflori). Its common name in English-speaking countries isFleeceflower Root and is known in China as Heshouwu, Shouwu, orChishouwu.

Heshouwu can be harvested in autumn and winter when leaves wither,washed clean, and the large one cut into pieces, and then dried toproduce a dried heshouwu. Heshouwu can also be prepared by steaming(e.g., for 3 hr) to produce a steamed heshouwu, optionally in thepresence of wine to produced the so-called wine-processed heshouwu. Theslices or pieces of heshouwu may be mixed with thoroughly with blackbean juice and stewed in a suitable non-ferrous container until thejuice is exhausted. The mixture is dried to solidify and then cut intoslices to produce the so-called prepared heshouwu.

Crude heshouwu and prepared heshouwu may differ in the composition. Itis known that all kinds of heshouwu contain free phosphatidylcholine(lecithin), phosphatidylinositol, phosphatidylcholine,phosphatidylethanolamine (cephalin), N-free phosphatidylethanolamine andsphingolipids. Crude heshouwu usually contains 3.7% phospholipids, andhigher than processed heshouwu. Heshouwu also contains emodins such asanthraquinones or anthrones which mainly glycoside with glucose andrhamnose to form mono- or di-glycoside, chrysophanol, emodin, rhein,chrysophanol ester, and chrysophanin acid anthrone. Processed heshouwuhas a lower concentration of anthraquinones. Heshouwu also containstetrahydroxystilbene glycoside and its analogues, and the processedheshouwu have slight higher concentration. Heshouwu is abundant of traceelements, such as calcium, iron, manganese, copper, and zinc at aconcentration of about 421 ug/g, tens times higher than most herb. Inaddition, heshouwu has high concentration of starch, soluble amylose,vitamins, amino acids, and coarse fat.

Also optionally, the culture medium may further comprise various aminoacids, preferably all 20 natural amino acids (e.g., alanine,asparagines, aspartic acid, cysteine, glutamic acid, glutamine, glycine,phenylalanine, histidine, isoleucine, lysine, leucine, methionine,proline, arginine, serine, threonine, valine, tryptophan, and tyrosine),for providing nutrition support to cell growth. The amino acids may bechemically synthesized or obtained from natural sources. For example, afull spectrum of natural amino acids may be obtained by extractingearthworms, a rich source of protein/amino acids, in oil or alcohol.

The culture medium may further comprise nucleic acid bases such asadenine, cytidine, guanine, thymine and uridine.

In a particular embodiment, the oil in the culture medium is anoil-extract of earthworm wherein the amount of earthworm is 2–60% byweight based on the total weight of the oil.

The tissue culture medium may further include a regular tissue culturemedium such as DMEM, MEM, etc.

The tissue culture medium may further comprise some or all of thecompositions of a typical medium suitable for the cultivation ofmammalian cells. Examples of these reagents for tissue culture include,but are not limited to, a) Amino acids such as arginine, cystine,glutamine, histidine, isoleusine, leucine, lysine, methionine,phenylalanine, threonine, tryptophan, tyrosine, and valine; b) Vitaminssuch as biotin, choline, folate, nicotinamide, pantothenate, pyridoxal,thiamine, and riboflavin; c) Salts such as NaCl, KCl, NaH2PO4, NaHCO3,CaCl2, and MgCl2, d) Proteins such as insulin, transferrin, specificgrowth factors; and e) Miscellaneous: glucose, penicillin, streptomycin,phenol red, whole serum.

In yet another aspect of the invention, isolated potentiallyregenerative cells from a predetermined site of the body of a livemammal are provided. The isolated potentially regenerative cells, whencultured in a culture medium under suitable conditions, are capable ofbeing activated to continuously proliferate and differentiate to form atissue-organ which shares substantially the same physiological structureand at least one physiological function with that of the correspondingtissue in situ and in vivo.

The potentially regenerative cells may be isolated from any site of thebody of the mammal such as an adult body of a human, for example, thebrain, heart, liver, lung, intestine, stomach, kidney, bone marrow, andskin. The isolated potentially regenerative cells are not embryonic stemcells, and are not from the blastocyst of the mammal.

The culture medium may comprise at least 50% of water and a sterolcompound that is dissolved in a fatty acid-containing oil at aconcentration at least 0.1% by weight based on the weight of the oil andadded to the water. The sterol compound preferably forms ester with thefatty acid in the oil under suitable conditions such as at hightemperatures (e.g., >100° C.).

The isolated potentially regenerative cells, when cultured in theculture medium, may be able to continuously proliferate anddifferentiate for at least 5 days, preferably for at least 10 days, morepreferably for at least 30 days, and most preferably for at least 50days.

The compositions and methods of the present invention can be utilized togenerate in vitro a large amount of regenerative cells, tissues and/ororgans with normal physiological structure and function. Thesebiological materials can serve as extremely valuable models for basicscientific investigation in every aspect of life sciences, and beutilized in many practical applications such as nutraceutical discovery,drug screening, pharmacokinetics studies, medical devices andtissue/organ transplantation.

EXAMPLES Example 1 Culture of Human Intestinal Cells in Vitro

An in vitro experiment on human intestinal cells was carried out byfollowing this protocol:

Obtained normal human living intestine from surgical operations, firstput the tissue lump into double antibiotics (penicillin andstreptomycin)-containing, precooled phosphate buffered saline (PBS),rinsed three times, cut the large tissue lump into small pieces of 1mm³, then rinsed them with same PBS two times, put these washed smallpieces into 0.25% trypsin or 1% collagenase digestive solution preparedwith sterile PBS, digested them under the condition of 4° C. overnightwith shaking (about 16 hours) or 37° C. water bath, 3 hours withshaking.

Used pipette or aspirator to blow and aspirate the tissue repeatedly orpoured the mixture of tissue pieces with enzyme solution into stainlesssteel filter, and used syringe plug to grind the tissue pieces until allpieces were dispersed into single cells.

Seated still the mixture of single cells and enzyme solution (e.g.trypsin or collagenase, known to skilled artisans in this field) for 5minutes. Discarded the undigested and precipitated large pieces andindigestible connective tissues, moved the supernatant containing largeamount of cells and digestive enzyme to another centrifuge tube, and ifnecessary, filtered the supernatant with stainless steel filter.

Centrifuged the supernatant at 4° C., 1500 rotations per minute (rpm)for 5 min, discarded the supernatant containing digestive enzyme, addedsmall amount of PBS, vortexed, and then added more precooled PBS andmixed.

Centrifuged the supernatant at 4° C., 1500 rpm for 5 min, discarded thesupernatant, added small quantified amount of precooled PBS, vortexedand added more quantified amount of precooled PBS, mixed and counted thecell number.

Centrifuged the supernatant at 4° C., 1500 rpm for 5 min, discarded thesupernatant, added small quantified amount of 15% newborn calfserum(NCS) MEM medium or RPMI 1640 medium, vortexed and added moreappropriately quantified amount of medium, adjusted cell concentrationto 1×10⁵ cells/ml, mixed.

Dispensed the cell suspension into wells of multi-well plates (96 wells,24 wells, 12 wells or 6 wells) precoated with rat tail collagen. 200 μlper well for 96-well plate, 1 ml for 24-well plate, 2 ml for 12-wellplate, and 4 ml for 6-well plates.

Cultured the cells in 37° C., 5% CO₂ incubator or in 37° C., 5% CO₂ and45% O₂ incubator. 24 hours later, all the cells attached to well bottomand grew well. Divided the wells into two groups: test group and controlgroup. In test group wells, 15% fetal calf serum (FCS) MEM medium or 15%FCS RPMI 1640 medium plus sitosterol (0.2% w/w) was added.

Choice of regular tissue culture medium (e.g. MEM, DMEM, RPMI) is knownto skilled artisans in the art. Only regular tissue culture medium wasadded into the control group wells. In the test group, the tissueculture medium contains 10 grams of the inventive cell growth regulatorper 100 ml medium.

Changed the medium according to the routine protocol, i.e., discardedhalf of the old medium and added same amount of fresh medium, e.g., 15%FCS MEM medium or 15% FCS RPMI 1640 medium, from then on, changed themedium every three days, observed cells regularly.

After 55 days of culture, as in FIG. 1, mucosal tissue cells appeared indifferent forms, all cells lived vigorously in the medium and some weredividing, In these live cells, some proliferated persistently and aretermed herein potentially regenerative cells (PRCs), but some did nothave these characteristics.

Refer to FIG. 2. The large dividing cells were from PRCs of replicatingtissues and organs, the small, non-dividing cells were non-dividing,preexisting PRCs, and newly created PRCs. These PRCs proliferatedcontinuously in the medium under the effect of the inventive cell growthregulator, manifesting typical characteristics of stem cells. Afterexamination of cellular function, they can be used as in vitroexperimental models for studies on normal cell structure and function.

Refer to FIG. 3. Some of the persistently proliferating cells in themedium began to link and form tissues, the cells in the newly formedtissues changed from a round form to a tissue-specific form. Some of thecells still proliferated continuously. The tissues they formed can beused as in vitro experimental models for studies on normal tissuestructure and function.

Refer to FIG. 4. After proliferating cells linked and formed primarytissues, the latter continued to assemble into fully developed tissues,e.g., villi of intestinal mucosa, following the predetermined geneticprogram of the PRCs.

In the course of above-mentioned research in which cells evolved intotissues and organs in vitro, the inventor investigated the source of thecells with the stem cell-like proliferating ability. It was observedunder microscope that some of the growing single cells began to divide,become terminally differentiated cells, and did not form new tissues,whereas other cells proliferated persistently and formed new tissueswith different forms, and several of different forms of tissuesassembled and formed large tissues and organs.

To find out the reason, the inventor fluorescently labeled both thecells with proliferation potential and the proliferating cells derivedfrom asymmetrical division of proliferating cells. The results indicatedthat the cells in both groups shared identical markers, suggesting thatthe source cells of tissue regeneration may be the non-proliferatingcells derived from the asymmetrical division of proliferating cells.These non-proliferating cells are termed “Potentially RegenerativeCells” (PRCs). These cells might be duplicates of cells left over inevery stage of development and tissue regeneration; and they carried allof the information specific to that stage. Together with tissues cellsdirectly from the cell proliferation, PRCs formed tissues or organs andappear to be the same as regular tissue cells morphologically.

However, when adult tissues or organs are injured, dysfunctional ordegenerated, the PRCs are activated, divide and proliferate in situ andform new tissues and organs to compensate for the functional andstructural defects. The developmental process of PRCs is illustrated inFIG. 5.

Example 2 Culture of Mouse Intestinal Mucosal Villi in Vitro

The in vitro experiment on mouse intestinal cells was carried out byfollowing this protocol:

Killed Kunming mouse provided by qualified Laboratory Animal Institute,Chinese Academy of Medical Sciences with routine protocol known toskilled artisans in this field. Sterilized its body surface two timeswith 75% ethanol, 5 minutes each time. According to anatomicallocalization, took the exact living tissues. To disperse the tissuesinto single cells, put the tissue pieces into double antibiotics(penicillin and streptomycin)-containing, precooled PBS, rinsed threetimes, cut the large tissue lump into small pieces of 1 mm³, then rinsedthem with the same PBS two times, put these washed small pieces into0.25% trypsin or 1% collagenase digestive solution prepared with sterilePBS, digested them under the condition of 4° C. overnight with shaking(about 16 hours) or 37° C. water bath, 3 hours with shaking.

Cultured with the same method as in Example 1, the difference was thatsmall explants of proximal intestine from fetus Kunming mice were usedin this example. In the test group, 5 to 10 explants were planted ineach multi-well plate containing the inventive culture medium. Culturedthe attached explants with an interval of 1 mm between two explants.According to total weight of medium in each well, sitosterol of 1%(w/w), beeswax of 5% (w/w), and optionally propolis of 5% (w/w) could beadded as cell growth regulators based on the total weight of the cellgrowth regulator. The concentration of cell growth regulators was of 20grams per 100 ml medium.

The same explants were used in the control group, but no above-mentionedcell growth regulator was added in control wells. Other conditions werethe same as in test group. Cultured both groups according to routineprotocols.

Refer to FIG. 6. Through continuous culture for 30 days, explants intest group attached to the well bottom and grew very well, but those incontrol group began to detach from the bottom of the wells.

Refer to FIG. 7. Through continuous culture for 60 days, in the testgroup, the intestinal explants continued to live, cells appeared to beseparated from the tissues. Single cells were observed to suspend in themedium. In contrast, in the control group, intestinal explants began todegenerate and die and the isolated cells in a small number also beganto die. With continuous culture in the inventive culture medium, thesingle cells in the test group began to form clones(FIG. 8).

Refer to FIG. 9. Continued to culture intestinal cells in the testgroup. It was observed that the cells began to aggregate and adhere toeach other. These connected cells formed primary tissues, and the latterexpanded and formed intestinal mucosal tissues.

Refer to FIG. 10 showing the ends of intestinal villi which was formedat the last stage of the development. The amplified intestinal mucosawere also shown.

Through above-mentioned culture process, single cells were observed tomigrate from tissue explants and into the surrounding medium (FIG. 11A).A large number of intestinal single cells in different forms appear inthe culture, and these single cells continued to proliferate and beganto form primary tissues (FIG. 11B). These primary tissues aggregatedstep by step, and integrated with each other to form tissues with basicfunctions (FIG. 12 a). As shown in FIG. 12A, it is obvious that therewere cell-cell adhesion, tissue-tissue connection and tissue movementfor connection. In FIG. 12B, formation of tissues with physiologicalstructures could be found, including structures resembling thecross-sections of the intestinal villus bases, cells linking with eachother to form a circle, and scattered cells approaching these tissues tosurround the villi.

Refer to FIG. 13. Through culturing, intestinal cells from mouseexplants began to form the villus organ. It can be seen that the cellsreplicated along the basic circle of villi, eventually formed newintestinal villi and completed the cloning of intestinal mucosa invitro.

Refer to FIG. 14 that compares cross-sections of the intestinal villiregenerated in vitro in the present invention and with the one shown inYang, Q. et al. (2001) Science 294:2155–8). It is very obvious that atleast morphologically the in vitro generated intestinal villi have thesame types of cells as the ones identified in a tissue biopsy in thispublished literature: epithelial cells, goblet cells, Paneth cells, andendocrinal cells.

FIG. 15 shows the comparison of the vertical section of normal villi ina tissue section in a biopsy and the one regenerated in vitro accordingto the present invention. As shown in FIG. 15, the in vitro generatedvilli have the same morphology and structure as the ones in the biopsy.

Example 3 Culture of Mouse Bone Marrow Tissue in Vitro

The in vitro experiment on mouse bone marrow was carried out byfollowing this protocol:

Harvested bone marrow cells from Balb/c mouse provided by qualifiedLaboratory Animal Institute, Chinese Academy of Medical Sciences. Thecell collection method is known to skilled artisans in this field. Inthe test wells, RPMI1640 medium and a mixture of stigmasterol,β-sitosterol and campesterol (1:1:1 in weight, about 1% w/w of the totalweight of the medium), beeswax at 10% (w/w medium), and obabenine at0.003% (w/w medium).

After continuous culture of the bone marrow for 64 days, the followingresults were obtained. Refer to FIG. 16A. Progenitor cells appeared inthe test group, these cells aggregated and formed large and smallcolonies, and evolved into bone marrow tissues gradually.

Refer to FIG. 16B, after 10 days of continuous culture, bone marrowprogenitor cells appeared in the control group. However, after 15th dayof culture, the number of fibroblasts increased gradually and no bonemarrow tissues formed.

Example 4 Culture of Rat Nerve Tissue in Vitro

An in vitro experiment on rat bone neurons was carried out by followingthis protocol:

Collected neurons from SD rats provided by qualified Laboratory AnimalInstitute, Chinese Academy of Medical Sciences. The neuron collectionmethod was known to skilled artisans in this field. In the test wells,the growth medium was L15 medium plus an inventive cell growth regulator(15 g/100 ml medium) which is mixture of stigmasterol, β-sitosterol andcampesterol (1:1:1 in weight, about 1% w/w of the total weight of themedium), beeswax of 10% (w/w medium), baicalin of 1% (w/w medium), andberberine of 0.001% (w/w medium). The control well only contained L15medium.

After continuous culture for 25 days, the following results wereobtained. Refer to FIG. 17A. there was obvious elongation of nervetissue in test group 1. In contrast, nerve tissue contracted anddegenerated in control group 2 (upper pictures in FIG. 17A). Observedmicroscopically under ×250 magnification, the regenerated nerve tissuein test group 1 appeared with clear grains and in a form of bundle. Incontrast, nerve tissue in control group 1 showed obvious degeneration(upper pictures in FIG. 17B). HE staining indicated the same results intest group 2 and test group 1 (lower pictures in FIG. 17B) while nervetissue in control group 2 showed obvious degeneration (lower pictures inFIG. 17B).

Example 5 Culture of Mouse Pancreatic Cells in Vitro

An in vitro experiment on mouse pancreatic cells was carried out byfollowing this protocol:

Collected pancreatic cells from Kunming mouse provided by qualifiedLaboratory Animal Institute, Chinese Academy of Medical Sciences. Thecell collection method was known to skilled artisans in this field. Inthe test wells, In the test wells, the growth medium was Ham's F12medium plus an inventive cell growth regulator (50 g/100 ml medium)which is a mixture of spinasterol, 24-dehydrocholesterol, poriferasteroland daucosterol (1:1:1:1 in weight, about 20% w/w of the total weight ofthe medium), beeswax at 0.1% (w/w medium), baicalin at 1% (w/w medium),berberine at 0.001% (w/w medium), and narcotoline at 0.001% (w/wmedium). The control medium only contained Ham's F12 medium.

Continuously cultured the cells for 40 days. Refer to FIG. 18,pancreatic cells evolved into pancreatic tissues and the tissues furthermatured after 92 days of culture. In contrast, in the control group alarge number of cells died and no pancreatic tissues formed after 55days of culture. The pancreatic cells were necrotic and died extensivelyin the control group.

To verify the function of pancreatic tissues in the test group, themedium was collected from the wells where the pancreatic cells werecultured for at least 60 days and examined for the levels of theamylopsin and insulin in the medium by using a method known to skilledartisans in the field. The concentration of amylopsin was 14.6 unit/L inthe test group and more than 850 unit/L in the control group (leftpanel, FIG. 19). The concentration of insulin was 0.15 μunit/ml in thetest group and 0.01 μunit/ml in the control group (right panel, FIG.19). The difference between two groups is statistically significant.

Example 6 Culture of Mouse Renal Cells in Vitro

An in vitro experiment on mouse renal cells was carried out by followingthis protocol:

Collected renal cells from Kunming mice provided by qualified LaboratoryAnimal Institute, Chinese Academy of Medical Sciences. The cellcollection method is known to skilled artisans in this field. In thetest wells, the growth medium was MB752/1 medium plus an inventive cellgrowth regulator (30 g/100 ml medium) which is a mixture of sterol at0.5% w/w of the total weight of the medium, beeswax at 20% (w/w medium),baicalin at 1% (w/w medium), berberine at 0.001% (w/w medium),narcotoline at 0.001% (w/w medium), and earth worm at 2% (w/w medium).The control medium only contained MB752/1 medium.

Refer to FIG. 20. Through continuous culture of renal cortical cells for60 days, new nephrons evolved in the test group. These nephrons werevery obvious. In contrast, a large number of cells died out in thecontrol group.

Example 7 Culture of Human Hair Follicles in Vitro

An in vitro experiment on human hair follicles was carried out byfollowing this protocol:

Collected hair follicles through depilation of human head hair and bodyhair. Obtained the follicular cells from the follicular bulge area. Inthe test wells (24-well plate), the growth medium was 5% FCS MEM medium(2 ml) plus an inventive cell growth regulator (35 g/100 ml medium)which is a mixture of β-sitosterol at 0.5% w/w of the total weight ofthe medium, beeswax at 20% (w/w medium), baicalin at 1% (w/w medium),berberine at 0.001% (w/w medium), narcotoline at 0.001% (w/w medium),and earth worm at 0.001% (w/w medium). The control medium only contained5% FCS MEM medium.

Follicular cells showed obvious colonization through continuous culturefor 70 days. After 78 days of culture, follicular cells attached to eachother, formed follicles and further evolved into follicular tissues andtissue-organs (FIG. 21A). Eventually, hair grew out the follicles (FIG.21B).

Example 8 Culture of Rat Cardiomuscular Cells in Vitro

An in vitro experiment on rat cardiomuscular cells was carried out byfollowing this protocol:

Collected cardiomuscular cells from SD rat provided by qualifiedLaboratory Animal Institute, Chinese Academy of Medical Sciences. Thecell collection method is known to skilled artisans in this field. Inthe test wells, the growth medium was CMRL1066 medium plus an inventivecell growth regulator (25 g/100 ml medium) which is a mixture of sterolat 6% w/w of the total weight of the medium, beeswax at 20% (w/wmedium), baicalin at 10% (w/w medium), obabenine at 0.02% (w/w medium),berberine at 0.01% (w/w medium), narcotoline at 0.01% (w/w medium), andearth worm at 2% (w/w medium). The control medium only containedCMRL1066 medium.

Refer to FIG. 22. Through continuous culture for 48 days, thecardiomuscular cells began to link and cardiomuscular tissues formedafter culture for 65 days.

Example 9 Culture of Rat Thymocytes in Vitro

An in vitro experiment on rat thymocytes was carried out by followingthis protocol:

Collected thymocytes from Wistar rat provided by qualified LaboratoryAnimal Institute, Chinese Academy of Medical Sciences. The cellcollection method is known to skilled artisans in this field. In thetest wells, the growth medium was CMRL1066 medium plus an inventive cellgrowth regulator (40 g/100 ml medium) which is a mixture ofstigmasterol, β-sitosterol, chalinosterol, and γ-sitosterol(0.5:1:0.85:0.5, 10% w/w of the total weight of the medium), beeswax at15% (w/w medium), baicalin at 2% (w/w medium), obabenine at 0.05% (w/wmedium), berberine at 0.03% (w/w medium), and earth worm at 0.01% (w/wmedium).

Refer to FIG. 23. In the test group, thymocytes began to aggregate andconnect after continuous culture for 15 days, and the replication ofthymic tissues completed after continuous culture for 34 days. Incontrast, thymocytes in the control group began to die after 8 to 10days of culture, and eventually no thymic tissues formed.

Example 10 Culture of Rat Hepatocytes in Vitro

An in vitro experiment on rat liver cells was carried out by followingthis protocol:

Collected hepatocytes from Wistar rats provided by qualified LaboratoryAnimal Institute, Chinese Academy of Medical Sciences. The cellcollection method is known to skilled artisans in this field. In thetest wells, the growth medium was 15% FCS CMRL1066 medium plus aninventive cell growth regulator (50 g/100 ml medium) which is a mixtureof stigmasterol, β-sitosterol, chalinosterol, and γ-sitosterol(0.5:1:0.85:0.5%, 10% w/w of the total weight of the medium), beeswax at10% (w/w medium), baicalin at 1% (w/w medium), obabenine at 0.05% (w/wmedium), berberine at 0.03% (w/w medium), and earth worm at 0.01% (w/wmedium).

Refer to FIG. 24, the liver cells began to proliferate, aggregate andlink after continuous culture for 15 days. After culture for 25 days,hepatic lobules appeared and the liver tissue replication completed. Incontrast, liver cells in control group began to die after 8 to 10 daysof culture and eventually no intact liver tissue formed.

Example 11 Regeneration of Mouse Stomach in Vivo and in Situ

In this example, a mouse model for with acute hemorrhagic gastric ulcerwas created by i.g. with ethanol. Afterwards stomachs of mice in thetest group were filled with the same cell growth regulator as used inexample 2. Three day later, the mice of test and control groups weresacrificed and their stomachs isolated.

FIG. 25 compares the stomachs of the mice in the test and controlgroups. It is obvious that mucosa damaged by the acute gastric ulcer inthe test group were repaired without any scar. In contrast, in thecontrol group, typical hemorrhagic ulcer of mucosa occurred. As shown inthe left panel of FIG. 25, the black spots were ulcers and necrosis ofmucosa appeared.

Example 12 Regeneration of Human Stomach in Vivo and in Situ

FIG. 26 shows a stomach of a patient with gastroduodenal ulcer, anobvious ulcer could be observed in mucosa of the gastric angular areawith mucosa necrotized in omnilayer and peripheral tissues appearinginflammatory. After treatment for ten days with the same cell growthregulator as used in example 2, gastric mucosa in the lesion area wererepaired in vivo and in situ with no scar formation as observed bystomachoscopy. The same results were obtained for the peripheraltissues.

Example 13 Regeneration of Plant Tissue in Vivo and in Situ

In this example, a growing winter melon was chosen. Three pieces of thinrinds in 2 cm×2 cm were scraped off with a knife and the wounds on themelon were treated with three different methods within 5 minutes: onesmeared with vegetable oil (sesame oil or soy oil), one coated withwater-wetted gauze, and the third with nothing. The treatments lastedfor ten days, one time per day.

Ten days later, new rind grew in the wound which was smeared withvegetable oil. In contrast, the wound which was coated with water-wettedgauze was rotten, and there was a “scar” in wound treated with nothing.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative and are not to be taken aslimitations upon the scope of the invention, which is defined solely bythe appended claims and their equivalents. Various changes andmodifications to the disclosed embodiments will be apparent to thoseskilled in the art. Such changes and modifications, including withoutlimitation those relating to the substituents, means of preparationand/or methods of use of the invention, may be made without departingfrom the spirit and scope thereof.

1. A method for culturing cells in vitro, comprising: providing tissuecells or a tissue isolated from a predetermined site of the body of amammal; forming a tissue culture medium by adding fatty acid-containingoil in which a sterol compound of at least 0.1%, baicalin of 0.001–2%and wax of 1–20% by weight based on the total weight of the oil aredissolved to a culture medium containing at least 50% of water; andculturing the isolated tissue cells or tissue in the tissue culturemedium.
 2. The method of claim 1, wherein the tissue cells or tissue areisolated from a rodent, a primate or a human.
 3. The method of claim 1,wherein the tissue cells or tissue are isolated from a fully developedadult human.
 4. The method of claim 1, wherein the tissue cells ortissue are isolated from live mammal.
 5. The method of claim 1, whereinthe isolated tissue cells or tissue are from the brain, heart, liver,lung, intestine, stomach, kidney, bone marrow, or skin of the mammal,excluding embryonic stem cells and the blastocyst of the mammal.
 6. Themethod of claim 1, wherein the tissue is processed in vitro to producecells which are then isolated and cultured in the tisuue culture mediumto produce the tissue-organ.
 7. The method of claim 1, wherein the cellscontained in the isolated tissue cells or tissue are activated in thetissue culture medium to continuously proliferate and differentiate forat least 5 days.
 8. The method of claim 1, wherein the cells containedin the isolated tissue cells or tissue are activated in the tissueculture medium to continuously proliferate and differentiate for atleast 30 days.
 9. The method of claim 1, wherein the cells contained inthe isolated tissue cells or tissue are activated in culture medium tocontinuously proliferate and differentiate for at least 50 days.
 10. Themethod of claim 1, wherein the oil comprises vegetable oil or animaloil.
 11. The method of claim 1, wherein the oil comprises an oilselected from the group consisting of corn oil, peanut oil, cottonseedoil, rice bran oil, safflower oil, tea tree oil, pine nut oil, macadamianut oil, camellia seed oil, rose hip oil, sesame oil, olive oil, soybeanoil and combinations thereof.
 12. The method of claim 1, wherein the oilcomprises sesame oil.
 13. The method of claim 1, wherein the fatty-acidis selected from the group consisting of palmitic acid, linoleic acid,oleic acid, trans-oleic acid, stearic acid, arachidic acid, andtetracosanoic acid.
 14. The method of claim 1, wherein the sterolcompound is an animal sterol or a plant sterol.
 15. The method of claim1, wherein the sterol compound is selected from the group consisting ofstigmasterol, campesterol, β-sitosterol, chalinosterol, clionasterol,brassicasterol, α-spinasterol, daucosterol, avenasterol, cycloartenol,desmosterol, and poriferasterol.
 16. The method of claim 1, wherein thesterol compound is a combination of stigmasterol, β-sitosterol, andcampesterol.
 17. The method of claim 1, wherein the sterol compound is acombination of stigmasterol and β-sitosterol.
 18. The method of claim 1,wherein the sterol compound is a combination of bras sicasterol andβ-sitosterol.
 19. The method of claim 1, wherein the sterol compound isa combination of brassicasterol, stigmasterol and β-sitosterol.
 20. Themethod of claim 1, wherein the wax is edible wax.
 21. The method ofclaim 1, wherein the wax is selected from the group consisting ofbeeswax, castorwax, glycowax, and carnaubawax.
 22. The method of claim1, wherein the wax is beeswax.
 23. The method of claim 1, wherein theconcentration of baicalin is about 0.02–0.5% by weight based on thetotal weight of the oil.